Ferment activator based on lactic acid bacteria and method for preparing a dairy product using same

ABSTRACT

The invention concerns a ferment activator based on lactic acid bacteria, characterised in that it comprises at least: a nitrogenous substance, a buffer system capable of maintaining the activity pH of the lactic acid bacteria with which said activator is to be associated at a value ranging between 5 and 7, and free of added sugar(s) capable of being metabolised by said lactic acid bacteria. The invention also concerns a method for preparing a dairy product characterised in that it consists in using said activator.

This application is a 371 of PCT/FR01/02928 filed Sep. 20, 2001.

The present invention relates to an activator for a ferment based onlactic acid bacteria, to the use of this activator for the preparationof milk products and to the method for preparing a milk productcharacterized by the use of this activator.

The fermentation of milk is generally carried out by inoculating it witha bacterial culture commonly designated by the name of starter orferment. This ferment generally contains anaerobic or microaerophilicbacteria belonging to the group of Gram-positive bacteria which fermentsugars into their respective acids. The acid mainly produced is lacticacid from lactose.

Generally, these ferments contain mesophilic organisms having an optimumgrowth temperature of between 25 and 35° C. and/or said thermophilicorganisms having an optimum growth temperature of between 35 and 45° C.

The organisms most widely used and which are present in ferments arethose belonging to the genera Lactococcus, Streptococcus, Lactobacillus,Leuconostoc, Pediococcus, Bifidobacterium and Brevilbacterium.

The specific organisms belonging to the mesophile group compriseLactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris,Lactococcus lactis subsp. lactis biovar. diacetylactis, Leuconostoccremoris, Leuconostoc mesenteroides subsp mesenteroides, Leuconostocmesenteroides subsp lactis, this list not being exhaustive.

These thermophilic type bacterial species are, inter alia, Streptococcusthermophilus, Lactobacillus casei, Lactobacillus paracasei,Lactobacillus helveticus, Lactobacillus delbrueckii subsp. Bulgaricus,Lactobacillus bulgaricus and Lactobacillus acidophilus.

These ferments are used in a concentrated form or in a dry form, that isto say in the form of a powder, for example a freeze-dried orspray-dried powder, in a liquid form, or in a frozen state.

These types of formulation have the double advantage of preserving theviability of the cultures over a long period and of being mostparticularly appropriate for direct inoculation, according to which theferment is directly introduced into the manufacturing milk.Advantageously, no preliminary culturing is found to be necessary beforeuse unlike the so-called semidirect inoculation.

Although the present invention can also be effectively applied tosemidirect inoculation, it is found to be most particularly advantageousfor the so-called direct inoculation for the following reason: when thebacteria are introduced during a direct inoculation, that is to say inthe form of a dry, liquid or frozen concentrate, they are notimmediately effective and they require a restoration of activity. Therestoration of activity of this type of ferment requires a lapse of timefor adaptation corresponding, on the one hand, to the revival of thebacterium packaged in its natural form and, on the other hand, to therestoration of its metabolic activity. More precisely, this adaptationtime successively comprises a first phase for rehydration and a secondso-called “latent” phase corresponding more particularly to the“reawakening” of the metabolic activity of the bacteria. It is duringthis second phase that cellular repair, adaptation of the enzymaticsystem to its biological environment and initiation of cell divisionoccur. Whereas the rehydration phase is practically immediate, thelatent phase may extend up to 3 hours, and is of course damaging for theindustrialist in terms of profitability.

The direct inoculation technique offers decisive advantages: immediateavailability of the ferments with a reduced hindrance, possibility ofpreparing complex mixtures of different species or strains in definedand constant proportions, increased regularity of performance comparedwith traditional ferments prepared at the sites of use, productioncarried out in specialist units where each stage of the method isoptimized and controlled, the quality of the ferments vigorouslydefined.

The objective of the present invention is precisely to provide a meansfor significantly reducing this latent period.

Unexpectedly, the inventors have demonstrated that the bringing intocontact of a ferment based on lactic acid bacteria and preferablyso-called direct contact, with an activator in accordance with theinvention, prior to its introduction into the milk medium to be treated,made it possible to significantly reduce said latent period.

The first subject of the present invention is therefore an activator fora ferment based on lactic acid bacteria.

Its second subject is the use of this activator to activate, in liquidmedium, a ferment based on lactic acid bacteria.

Another aspect of the present invention relates to a ferment based onlactic acid bacteria thus activated.

Finally, the fourth subject of the present invention is a method forpreparing a milk product, characterized by the use of this activator orof an activated ferment according to the invention.

More precisely, the present invention relates to an activator for aferment based on lactic acid bacteria, characterized in that itcomprises at least:

-   -   a nitrogenous substance,    -   a buffer system capable of maintaining the pH for activity of        the lactic acid bacteria with which said activator has to be        combined at a value between 5 and 7,

and which is free of added sugar(s) which can be metabolized by saidlactic acid bacteria.

The claimed activator is particularly advantageous in terms of stabilityand/or of gain in productivity, of a ferment for direct inoculation inliquid form.

Accordingly, because of the absence, from the activator, ofmetabolizable sugar(s), no substantial production of lactic acid whichwould be damaging to the stability of the lactic acid bacteria isinitiated during the bringing of this activator into contact with theferment to be activated. Greater stability over time of the activatedferment follows.

Consequently, the joint use of the activator with a ferment based onlactic acid bacteria advantageously makes it possible to preserve andstandardize the metabolic activity of the activated bacteria over aprolonged period of time compared with that observed with an identicalferment in a nonactivated form.

Furthermore, quite advantageously, the use of the activator with aferment makes it possible to delay cell multiplication or quite simplyto limit cell multiplication, while allowing the ferments to resumetheir metabolic activity and while maintaining the activated fermentaccording to the invention effective. This is illustrated by example 3.

An activated ferment according to the invention is advantageouslyeffective over a period extending up to 72 hours, more particularly overa period extending up to 48 hours, preferably over a period extending upto 24 hours.

Accordingly, a ferment based on an activated Lactococcus lactisaccording to the invention is effective over a period extending up to 72hours whereas an identical, but nonactivated, ferment manifests asignificant loss of activity above 3 hours.

Moreover, the inventors have observed that the presence of the activatorwas advantageous in terms of equilibrium of microbial populations in theactivated system. This is in particular illustrated in example 3presented below.

As regards gain in productivity, it is mainly linked to the reduction ofthe so-called “latent” period.

More precisely, for the purposes of the present invention, theexpression “latent period” designates the period elapsing between themoment of introducing the activated or nonactivated ferment into a milkproduct and the moment where the metabolic activity of the lactic acidbacteria present in this ferment is verified by a significant reductionof the pH of the milk medium due to the formation of lactic acid. Thisso-called significant reduction in the pH is in fact an arbitrary valuewhich depends on the measuring apparatus selected. However, as a guideand in the case of the apparatus used in the examples illustrating theinvention, this reduction in the pH is evaluated at about 0.08. Moregenerally, it can be estimated that this significant reduction isreached when the pH of the milk medium treated has decreased by about 5%of its initial value.

This gain in productivity is particularly significant for ferments basedon lactic acid bacteria comprising totally or at the very leastpredominantly mesophilic-type bacteria. Advantageously, the combinationof an activator with a ferment based on mesophilic-type lactic acidbacteria reduces the latent period by about 10 to 25% of its standardvalue.

Consequently, and as is evident from the examples presented below, aferment based on lactic acid bacteria in a freeze-dried form, mixedprior to its introduction into the milk with an activator according tothe invention, restores an acidifying power in the milk much morerapidly compared with the standard ferment, that is to say in anonactivated form.

The nitrogenous substances present in the claimed activator are orresult preferably from nitrogenous substances of the peptide and aminoacid type and/or from one or more dairy or nondairy proteins.

By way of representatives of the proteins suitable for the invention,there may be mentioned in particular β-lactoglobulin, albumin andalpha-lactalbumin, caseins and derivatives such as lactic casein, rennetcasein and caseinates, kappa-casein and beta-casein.

As other examples of nitrogenous substances, there may be mentioned inparticular yeast extracts and more preferably an extract of the yeastSaccharomyces cerevisiae, which may be combined with the proteins citedabove.

This fraction with nitrogenous substances constitutes about 50 to 90%,and preferably 60 to 80% by weight of the activator.

The activator according to the invention does not contain addedsugar(s), which means that there cannot be other sources of addedsugar(s) in this activator other than the nitrogenous substances.

It is indeed not excluded that these nitrogenous substances may containa certain quantity of metabolizable sugar(s), according to the source ofnitrogenous substances used.

As regards the buffer medium, its main role is to stabilize the pH ofthe activated ferment at a value close to between 5 and 7 during theperiod for its reactivation. Its presence proves particularlyadvantageous when it is intended to be combined with a ferment mainlycomprising mesophilic and thermophilic type lactic acid bacteria.

By way of illustration of the buffer mixtures which may be suitable forthe invention, there may be mentioned in particular those comprisingsalts such as magnesium and calcium salts as well as carbonate,phosphate and citrate salts.

They are preferably a mixture of carbonates and more preferably amixture of calcium carbonate and magnesium carbonate.

According to a variant of the invention, nutritive elements which areuseful for maintaining the metabolic activity of the lactic acidbacteria are also combined with the nitrogenous substances and thebuffer mixture.

These nutritive elements generally include vitamins.

Likewise, cofactors useful for activating glycolysis may be present inthe claimed activator. As representative of these cofactors, there maybe mentioned in particular the inorganic salts Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺and Zn²⁺. They are generally used in an amount of 0.1 to 2%.

It is also possible to envisage incorporating into the activatortexturing agents of the hydrocolloid type, such as xanthan gum, guargum, and the like.

More precisely, the activator according to the invention is free ofadded sugar(s) which can be metabolized by said lactic acid bacteria.

Still more precisely, the activator according to the invention comprisesat most 15% by weight of sugars which can be metabolized by said lacticacid bacteria, preferably at most 10% of said sugars, and moreparticularly at most 5% of said sugars. It is understood that theyinclude sugar(s) not added in the sense defined above.

By way of illustration of the claimed activators, there may be mentionedmore particularly those comprising at least calcium caseinate, in anamount of 20 to 40% by weight, and as buffer mixture, a mixture ofcalcium carbonates and of magnesium carbonates. Yeast extracts andmanganese sulfate are also preferably present in this activator.

The claimed activator may be obtained by simply mixing its componentsand is generally present in a dry, generally pulverulent, form. However,it is also possible to envisage formulating it in a freeze-dried orfrozen form.

The claimed activator may also be provided in liquid form.

According to a preferred variant of the invention, the claimed activatoris provided in a sterilized form and is used while preserving thissterile aspect.

The second subject of the present invention is the use of an activatorin accordance with the present invention for activating a ferment basedon lactic acid bacteria prior to or during the inoculation of a milkmedium.

The ferment to activator ratio is between 10% and 70% by dry weight,preferably 20% to 60% by dry weight.

The use of this activator to activate, in liquid medium, a ferment basedon lactic acid bacteria has the advantage of an on-line, automatable,continuous or batch and aseptic inoculation.

The subject of the invention is also an activated ferment based onlactic acid bacteria, characterized in that it combines an activator inaccordance with the invention with at least lactic acid bacteria.

In this instance, the claimed activator is used in a quantity such thatthese components, namely the nitrogenous substances and buffer mixture,are present in sufficient quantities for a significant activation of theferment based on lactic acid bacteria to be observed.

As a guide, it is used in a quantity such that its content ofnitrogenous substances is adjusted in an amount of about 160 to 300% byweight relative to the weight of lactic acid bacteria present in theferment to be activated, preferably about 160% to 250%.

The activator may be mixed with the ferment either beforehand or at thetime of its use. However, according to a preferred embodiment, prior toits use, it is rehydrated in the presence of an activator in accordancewith the present invention. Generally, this combination is carried outin a liquid medium, preferably water.

The activator is rehydrated such that the quantity of activator isbetween 5% and 20% by weight of aqueous suspension, preferably between7% and 15%.

The rehydration and consecutive activation may be carried out at atemperature between 10° C. and 20° C. and preferably with stirring, soas to optimize activation and homogenization over time. The activatedferment is then used as it is for the inoculation, preferably directinoculation, of a milk medium.

The lactic acid bacteria capable of being combined with an activator inaccordance with the invention include all the bacteria customarily usedfor the production of milk products.

As a guide for lactic acid bacteria, there may be mentioned the bacteriabelonging to the genera Lactococcus, Streptococcus, Lactobacillus,Leuconostoc and Pediococcus.

The bacteria used in the dairy sector which belong to the generaBifidobacterium, Propionibacterium and Brevibacterium are alsoconsidered as lactic acid bacteria.

They may also be microorganisms more particularly used for ripening andin particular used in the cheese industry. By way of representatives ofthis second type of microorganisms, there may be mentioned in particularPenicillium roqueforti, Penicillium candidum, Geotrichum candidum,Tourla kefir and Saccharomyces kefir and Kluyveryomyces lactis.

The fourth subject of the present invention is a method for preparing amilk product comprising:

-   -   (i) bringing a ferment based on lactic acid bacteria into        contact with an activator in accordance with the present        invention, so as to obtain a so-called activated ferment,    -   (ii) inoculating the milk medium to be treated, preferably milk,        with said ferment in an activated form, and    -   (iii) incubating said milk medium under conditions favorable to        the metabolic activity of the lactic acid bacteria, so as to        obtain the expected milk product.

For the purposes of the present invention, the ferment obtained afterthe first stage (i) is so-called activated since compared with itsstandard form, that is to say not combined with an activator accordingto the invention, it manifests an improved bacterial activity. Thisimprovement manifests itself in terms of stability and gain inproductivity as discussed above.

As regards the preliminary stage (i), namely the bringing of the fermentinto contact with the claimed activator, it is generally carried outwithin a period sufficient for the production of the activated form andin a liquid medium. The corresponding suspension may be obtained byadding a liquid, preferably an aqueous medium, to the mixture of the twocomponents or by consecutive dispersion of the two components in saidliquid.

As specified above, the activator is used in a quantity such that itscontent of nitrogenous substances is adjusted to an amount of about 160to 300% by weight relative to the weight of lactic acid bacteria,preferably about 160% to 250%.

The use of the method according to the invention may be carried out bymeans of an inoculation device.

The preferred inoculation device, for carrying out the method accordingto the invention, is provided in the form of a sealed reservoir.

The sealed reservoir may be provided in the form of a closed pouchprovided with an internal stirring system and inlet and outlet means.

One of the inlet means allows the arrival of the aqueous medium in thesealed reservoir in order to carry out step (i). The aqueous medium issterilized beforehand, preferably it is filtered on a membrane of atmost 0.45 μm, more particularly at most 0.22 μm. It should be noted thattap water can be used.

The temperature of the aqueous medium on its arrival in the sealedreservoir is between 5° C. and 15° C., preferably between 8° C. and 12°C.

One of the other inlet means allows the arrival of gas into the sealedreservoir. The arrival of gas will allow the use of the internalstirring system of the receptacle.

The internal stirring system consists of a permeable internal pouch. Inthis case, the sealed reservoir comprises a permeable internal pouch anda closed external pouch. The stirring is carried out by successiveinjection of gas into the permeable internal pouch, which allows thetransfer of the suspension from the permeable internal pouch to theclosed external pouch.

A gas is advantageously used which is not involved in respiration and/oroxidation in the microorganisms, the ferments and the bacteria.

The injected gas is a chemically and biologically inert gas, preferablyargon, more particularly nitrogen or carbon dioxide, is injected.

The expression biologically inert gas is understood to mean a gas whichis not involved in the multiplication and degradation of themicroorganisms.

The gas pressure in the sealed reservoir, during the stirring, is lessthan 5 bar, preferably less than 1 bar.

The injection of gas can also be carried out over a regular timeinterval. Preferably, the gas is injected under pressure over a timeinterval of between 0.5 minute and 60 minutes.

The stirring allows the suspension of the ferments and of the activatorin the aqueous medium.

After stirring, the suspension of ferments and of the activator ismaintained in suspension by injection of gas according to the sameprinciple of successive injection of gas into the internal pouch.

The emptying of the sealed reservoir is carried out aseptically by theoutlet means, which makes it possible to carry out step (ii) of themethod.

This emptying is carried out by injecting gas inside the sealedreservoir, or by transferring the aqueous suspension of ferments and ofactivator using a pump or by gravity.

The inoculation of the milk medium to be treated with said ferment in anactivated form (step (ii)) is carried out at a flow rate of between 10ml/min and 1000 ml/min, preferably of between 100 ml/min and 500 ml/min.

Step (ii) according to the invention is carried out at a temperature ofbetween 5° C. and 40° C., preferably of between 10° C. and 15° C.

Step (ii) according to the invention is carried out over a periodextending up to 72 hours, more particularly over a period extending upto 48 hours, preferably over a period extending up to 24 hours.

Step (ii) may be carried out according to several variants.

A first variant of the method at the level of step (ii) consists ininoculating the milk medium to be treated once with said ferment in anactivated form. This is carried out by emptying the reservoir(s) in asingle operation. This involves a batch inoculation (a single reservoir)or a multi-batch inoculation (several reservoirs).

A second variant of the method at the level of step (ii) consists ininoculating the milk medium to be treated continuously with said fermentin an activated form.

A third variant of the method at the level of step (ii) consists ininoculating the milk medium to be treated batchwise with said ferment inan activated form.

The expression batchwise is understood to mean an inoculation cycleperformed in the following manner: the milk medium to be treated isinoculated over a lapse of time, then the inoculation is stopped, andthen the inoculation is resumed, this being for several cycles.

In the context of this third variant, the inoculation of the milk mediumto be treated with said ferment in an activated form (step (ii)) iscarried out at a flow rate of between 10 ml/min and 1000 ml/min,preferably between 100 ml/min and 500 ml/min, carried out for a regularor irregular time interval of between 1 minute and 600 minutes.

It should be noted that the sealed reservoir is advantageously attachedto a mobile station which can be moved over all the parts of theindustrial chain, before or after step (i) of the method according tothe invention.

The type of reservoir preferred for carrying out the method according tothe invention is of the disposable and/or sterile type.

This reservoir preferably consists of a flexible material such as, forexample, polypropylene, polyester, polyamide, cellulose or any otherflexible material compatible with food products, preferably it is madeof polyethylene.

The advantage of using the method according to the invention by means ofthe inoculation device as described above is to carry out a directinoculation, at room temperature, which is sterile, standardized andadaptable to each type of production and which ensures thebacteriological quality.

Another advantage of using the method according to the invention bymeans of the inoculation device as described above is to make theinoculation step of the lactic ferment simple and reliable.

The present invention also extends to the various forms for packagingthe claimed activator.

It is indeed possible to formulate the claimed activator in a packagingdistinct from that of the ferment based on lactic acid bacteria withwhich it is intended to be combined or, by contrast, to envisage acommon packaging in which the claimed activator and the ferment based onlactic acid bacteria are present, separately or otherwise.

This second packaging variant may in fact be designed so that it issuitable for mixing the ferment and the activator beforehand andtherefore for the preparation of the so-called activated ferment priorto the inoculation of a milk medium.

The examples given below are presented by way of illustration andwithout limiting the present invention.

Method

The lactic acid bacteria, alone or as a mixture, exhibit a greatdiversity of behavior. In the case of the present invention, theacidifying activity was selected as criterion for characterization.

The acidification of a milk medium occurs according to the followingchronological order:

-   -   inoculation of a milk (pH close to 6.6),    -   increase in the population of lactic acid bacteria by virtue of        the hydrolysis of the milk lactose,    -   production of lactic acid by the bacteria which results in a        decrease in the pH of the milk medium,    -   interruption of the growth of the bacteria which are gradually        inhibited by the lactic acid formed,    -   continuation of the production of acid up to a pH of 4.5.

The acidifying activity was assessed in the examples below using anautomated system for the monitoring and characterization of lacticferments by acquisition of measurement of pH in real time, alsodesignated below by the name CINAC.

CINAC is composed:

-   -   of Ingold type combination glass electrodes (24 channels for        measurements of pH placed in Erlenmeyer flasks containing the        inoculated medium and 8 channels for measurements of        temperature)    -   of a water bath regulated by a thermostat and in which the        Erlenmeyer flasks are placed    -   of an electronic card providing an analog signal and an        electronic interface converting the latter to a digital signal    -   of a PC microcomputer equipped with CINAC software offering the        following functions:    -   configuration of the system    -   data acquisition, treatment and storage    -   calibration of the probes at pH 7 and pH 4    -   calculation of the kinetic descriptors    -   graphical representations of the processed data    -   conversions of the data for the use of these data on other        software packages    -   programming of heat cycles in order to regulate the temperature        of the water bath    -   adjustment of the temperatures in order to correct variations in        the latter relative to the pH (this correction is made by means        of a PID regulator: proportional-integral-derivative)    -   execution of procedures for testing calibration data in order to        detect the dysfunctions linked to the probes.

CINAC processes the data by providing kinetics of acidification curvesand the descriptors of the latter.

The curves describing the kinetics represent variations in the pH and inthe rate of acidification (dpH/dt), as a function of time. They reflectvarious stages of growth: readaptation phase, acceleration, exponentialphase, slowing down, stationary phase.

The descriptors selected in examples for characterizing the kinetics ofacidification are:

-   -   Ta=latent period in min (time after which the pH varied 0.08 upH        below its initial value)    -   Vm=maximum rate of acidification in upH/min (rate taken at the        maximum of the absolute value of the derivative dpH/dt=f(t))    -   time 5.20=time to obtain a pH of 5.20 in minutes.

From all these parameters, it is possible to assess a gain or a loss inproductivity.

EXAMPLE 1 Preparation of a Rehydrated Concentrated Ferment According tothe Invention

Firstly, the activator according to the invention is prepared in asterile 1 l bottle containing a 45 mm double ring magnetic bar. Thevarious components of this mixture are presented in table I below:

TABLE I Products Quantity (g) Dairy proteins 30 Extract of S. cerivisiae35 Calcium carbonate 10 Magnesium carbonate 10 Manganese sulfate  5

The protein and mineral fractions constituting this mixture arepasteurized at 85° C. for 30 minutes and then they are mixed and thewhole is freeze-dried.

In the examples below, the activator thus obtained is then mixed with 50g of freeze-dried ferment and 870 g of sterile water. The dry mixture ispoured into water, with magnetic stirring, and the dissolution occurswithin a few minutes. 1 liter of a solution which contains 50 g offreeze-dried product is thus obtained.

The temperature for rehydration of the resulting mixture, namelyfreeze-dried product and claimed activator, is conducted according to aso-called “winter” heat cycle. This cycle restores the rise intemperature of a combination of 25 1 which starts at 15° C. and ends ata temperature of 20° C. which is reached in about 20 h.

EXAMPLE 2 Measurement of the Acidifying Activity of the LiquidConcentrate Obtained According to Example 1

The activity of the bacterial concentrate is assessed as a function ofthe storage time. It is measured after 20 minutes (considered as timeT0), 3 hours, 6 hours, 16 hours and 24 hours of storage.

The strains tested are predominantly mesophilic strains. They are moreprecisely the strains RA 024, RM 034 and MA 014 which are lacticferments marketed by RHODIA FOOD S.A.S.

The strain RA 024 is a mixture of Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris and Streptococcus salivarius subsp.thermophilus.

The strain MA 014 is a mixture of Lactococcus lactis subsp. lactis andLactococcus lactis subsp. cremoris.

The strain RM 034 is a mixture of Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactisbiovar diacetylactis and Streptococcus thermophilus.

The inoculation carrier used is semiskimmed milk at 30° C.

Because of the concentration, a dilution is made in order to be able toinoculate the acidification tests.

A control activity is started for each test carried out which uses 1 gof freeze-dried product in 200 ml of milk.

The controls are direct inoculations with nonactivated ferment inmanufacturing milk.

Because of the concentration of ferments used, a dilution of the productis carried out. Thus 1 g of ferment is dissolved in 200 ml of milk whichis used for the measurement of activity.

In the case of the rehydrated tests, a dilution is also carried out.

The inoculation should be carried out immediately so as not to penalizethe activity of the bacterial concentrate.

Measurement of the Acidifying Activity Over Time

The results obtained with each of the strains are presented in tablesII, III and IV below.

The data presented in these tables show the gains obtained in terms ofstability and productivity with the activated ferments according to theinvention compared with their respective nonactivated form.

TABLE II A 014 activated Test activated then stored Storage time 1 H 2 H4 H 6 H 8 H 24 h Time in min to have a 380 370 385 385 380 380 pH of5.20 MA 014 nonactivated control Time in min to have a 400 400 400 400400 400 pH of 5.20 Technological gain in 20 30 15 15 20 20 time inminutes obtained with the activated form

TABLE III RA 024 activated Test activated then stored Storage time 1 H 2H 4 H 6 H 8 H 12 H 24 h Time in min to have a 395 395 390 390 390 390395 pH of 5.20 RA 024 nonactivated control Time in min to have a 410 410410 410 410 410 410 pH of 5.20 Technological gain in 15 15 20 20 20 2015 time in minutes obtained with the activated form

TABLE IV RM 034 activated Test activated then stored Storage time 1 H 2H 4 H 6 H 8 H 12 H 24 h Time in min to have a 425 425 425 420 415 415415 pH of 5.20 RM 034 nonactivated control Time in min to have a 445 445445 445 445 445 445 pH of 5.20 Technological gain in 20 20 20 25 30 3030 time in minutes obtained with the activated form

EXAMPLE 3 Stability of the Microbial Populations in the Presence of anActivator in Accordance with the Invention

In this test, the stabilization of the populations is evaluated over aperiod of 24 hours in the rehydrated ferments RA 021, RA 022, RA 024 andRA 026 in the presence of the activator prepared according to example 1.

RA 021, RA 022 and RA 026 are strains comprising a mixture of mesophilicand thermophilic bacteria similar to that identified for the strain RA024 and are marketed by RHODIA FOOD S.A.S.

The conditions for mixing the ferment based on lactic acid bacteriaconsidered and the activator are identical to those presented in example2.

The results are presented in table V below.

TABLE V Commercial Group of Storage time mixtures strains T 0 4 h 00 8 h00 24 h 00 mesophile 3.10E + 10 3.30E + 10 3.50E + 10 3.20E + 10 RA021thermophile 5.10E + 09 5.00E + 09 5.40E + 09 6.50E + 09 mesophile3.10E + 10 3.00E + 10 3.00E + 10 3.00E + 10 RA022 thermophile 3.20E + 094.00E + 09 4.40E + 09 5.90E + 09 mesophile 3.20E + 10 3.50E + 10 3.00E +10 3.40E + 10 RA024 thermophile 4.90E + 09 5.30E + 09 5.80E + 09 6.70E +09 mesophile 2.40E + 10 2.60E + 10 2.50E + 10 2.20E + 10 RA026thermophile 3.70E + 09 4.20E + 09 4.00E + 09 4.00E + 09

The advantageous behavior of the activator toward the bacterialpopulation present in the ferment, and in particular the low cellmultiplication are evident from these results.

1. A method for the activation of a ferment based on lactic acidbacteria, prior to or during direct inoculation into a milk medium,comprising bringing said ferment into contact with an activator for saidferment which comprises at least: a nitrogenous substance, a buffersystem capable of maintaining the pH for activity of the lactic acidbacteria with which said activator has to be combined at a value between5 and 7, and which comprises at the most 15% by weight of sugars whichcan be metabolized by said lactic acid bacteria, the activator thusdelaying or limiting cell multiplication, while allowing the ferment toresume its metabolic activity.
 2. The method according to claim 1,wherein said activator is brought into contact with the ferment based onlactic acid bacteria in a liquid medium.
 3. A method for preparing amilk product comprising: (i) bringing a ferment comprising at leastlactic acid bacteria into contact witch an activator for said fermentwhich comprises at least: a nitrogenous substance, a buffer systemcapable of maintaining the pH for activity of the lactic acid bacteriawith which said activator has to be combined at a value between 5 and 7,and which comprises at the most 15% by weight of sugars which can bemetabolized by said lactic acid bacteria, the activator thus delaying orlimiting cell multiplication, while allowing the ferment to resume itsmetabolic activity, so as to obtain the ferment in an activated form,(ii) inoculating the milk medium to be treated with said ferment in anactivated form, and (iii) incubating said milk medium under conditionsfavorable to the metabolic activity of said lactic acid bacteria so asto obtain the expected milk product.
 4. The method as claimed in claim3, wherein the method may be carried out by means of an inoculationdevice.
 5. The method as claimed in claim 4, wherein the inoculationdevice is provided in the form of a sealed reservoir.
 6. The method asclaimed in claim 5, wherein the sealed reservoir may be provided in theform of a disposable reservoir and/or attached to a mobile station. 7.The method as claimed in claim 5, wherein the sealed reservoir may beprovided in the form of a pouch provided with an internal stirringsystem and inlet and outlet means.
 8. The method as claimed in claim 5,wherein one of the inlet means alilows the arrival of the aqueous mediumin the sealed reservoir in order to carry out step (i).
 9. The method asclaimed in claim 5, wherein the temperature of the aqueous medium on itsarrival in the sealed reservoir is between 50° C. and 150° C.
 10. Themethod as claimed in claim 5, wherein one of the other inlet meansallows the arrival of gas into the sealed reservoir.
 11. The methodaccording to claim 5, wherein the injected gas is a chemically andbiologically inert gas.
 12. The method as claimed in claim 5, whereinthe gas pressure in the sealed reservoir is less than 5 bar.
 13. Themethod as claimed in claim 5, wherein the injection of gas is carriedout between 0.5 minute and 60 minutes.
 14. The method as claimed inclaim 3, wherein step (ii) may be carried out according to severalvariants, either in batches, or in multi-batches, or continuously orbatchwise.
 15. The method as claimed in claim 3, wherein step (ii) iscarried out at a flow rate of between 10 ml/mm and 1,000 ml/mm.
 16. Themethod as claimed in claim 3, wherein step (ii) is carried out at atemperature of between 50° C. and 40° C.
 17. The method as claimed inclaim 3, wherein step (ii) is carried out over a period extending up to72 hours.
 18. The method as claimed in claim 3, wherein the fermentbased on lactic acid bacteria is brought into contact with saidactivator in a liquid medium.